Purification process for lipid-membrane-enclosed complex assemblages

Abstract

A chromatographic method for separation of lipid-membrane-enclosed compound assemblages (LMCAs) from chromatin heteroaggregates including degraded remnants from LMCAs in a sample, contacting the sample with a chromatographic material with a surface having cationic metal affinity ligands said chromatographic material being charged with ferric iron, manganese, calcium, or magnesium ions, and being equilibrated with an equilibration buffer, applying a gradient of a first elution buffer comprising a non-metal-chelating salt, thereby eluting LMCAs.

1 . A chromatographic method for separation of lipid-membrane-enclosed compound assemblages (LMCAs) from chromatin heteroaggregates including degraded remnants from LMCAs in a sample,
 contacting the sample with a chromatographic material with a surface having cationic metal affinity ligands said chromatographic material being charged with ferric iron, manganese, calcium, or magnesium ions, and being equilibrated with an equilibration buffer,   applying a gradient of a first elution buffer comprising a non-metal-chelating salt, thereby   eluting LMCAs.   
     
     
         2 . The method of  claim 1  wherein the LMCA is selected from the group consisting of a cellular vesicle, an extra-cellular vesicle, a non-cellular vesicle, a cellular organelle, an exosome, a small extra-cellular vesicle, a microvesicle, a medium extra-cellular vesicle, a ribosome, a mitochondrion, a lipid-enveloped virus or lipid-enveloped virus-like particle, an influenza virus, a corona virus, a lentivirus, a cytomegalovirus, a liposome and combinations thereof. 
     
     
         3 . The method of  claim 1  wherein the non-metal-chelating salt is a monovalent halide salt. 
     
     
         4 . The method of  claim 1  wherein the sample is selected from the group consisting of a cell culture harvest, a filtered cell culture harvest, a concentrated filtered cell culture harvest and a partially purified preparation containing a LMCA and chromatin heteroaggregates including LMCA remnants. 
     
     
         5 . The method of  claim 1  wherein chromatin heteroaggregates that include degraded remnants from LMCAs are eluted in a gradient of a second eluting buffer comprising a chelating salt. 
     
     
         6 . The method of  claim 5  wherein the chelating salt of the second eluting buffer is selected from the group consisting of a metal phosphate salt, a metal citrate salt, a metal salt of ethylenediaminetetraacetic acid (EDTA), a metal salt of ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and combinations thereof. 
     
     
         7 . The method of  claim 1  wherein the method is combined with at least one additional method for reduction of chromatin heteroaggregate content. 
     
     
         8 . The method of  claim 7  wherein the at least one additional method is a treatment with a solid phase bearing an anionic chromatography ligand. 
     
     
         9 . The method of  claim 8  wherein the solid phase bearing an anionic chromatography ligand is charged with iron or manganese ions. 
     
     
         10 . The method of  claim 1  wherein the sample is contacted with the chromatographic material and after an incubating time separated from the chromatographic material. 
     
     
         11 . The method of  claim 8  wherein the sample contains exosomes and is diafiltered by tangential flow filtration. 
     
     
         12 . The method of  claim 1  wherein the cationic metal affinity ligand is tris(2-aminoethyl)amine (TREN). 
     
     
         13 . The method of  claim 1  wherein the equilibration buffer comprises a pH value in the range of pH 5 to pH 9. 
     
     
         14 . The method of  claim 1  wherein the equilibration buffer has a conductivity value in the range of 1 mS/cm to 250 mS/cm. 
     
     
         15 . The method of  claim 7  wherein the at least one additional method is a treatment with nuclease enzymes. 
     
     
         16 . The method of  claim 11  wherein the sample is a cell culture harvest. 
     
     
         17 . The method of  claim 1  wherein the non-metal-chelating salt is sodium chloride. 
     
     
         18 . The method of  claim 1  wherein the equilibration buffer comprises a pH value in the range of pH 6.5 to pH 7.5. 
     
     
         19 . The method of  claim 1  wherein the equilibration buffer has a conductivity value in the range of 5 mS/cm to 10 mS/cm.